HER2 Testing: Past and Present
Transcript of HER2 Testing: Past and Present
Michael F. Press, M.D., Ph.D.
Professor
Harold E. Lee Chair for Cancer Research
Department of Pathology
Norris Comprehensive Cancer Center
University of Southern California
HER2 Testing: Past and Present(HER2 Testing in the Era of Changing Guidelines)
Faculty DisclosureCommercial Interest Nature of Relevant
Financial Relationship
Nature of Relevant Financial
Relationship
What was received For what role
Biocartis, SA Honorarium Scientific Advisory Board
Cepheid Research contract Investigator
Eli Lilly & Company Research contract
Honorarium
Investigator
Scientific Advisory Board
Zymeworks Research contract
Honorarium
Central Lab, Director
Scientific Advisory Board
Novartis Pharmaceuticals Research contract
Honorarium
Investigator
Scientific Advisory Board
Puma Biotechnology Research contract Honorarium
Investigator Consultant
HER2 Testing
• Background: HER2 / ERBB2 amplification is
directly correlated with HER2 overexpression in
frozen tissues.
• Comparisons of ASCO-CAP Guidelines for HER2
testing (2007, 2013 / 2014 and 2018) with IHC and
FISH.
• Summary of data for each ASCO-CAP FISH group
according to 2013 / 2014 and 2018 guidelines.
• Assessment issues with alternative control FISH
probes for HER2 “ISH-equivocal” breast cancers.
• Conclusion.
Correlation of HER2 Gene Amplification with Overexpression
Frozen
IHC
Amplification Level :
Northern
Western
10%27% 63% % Women
1> 1
0
5 -
10
2 -
5
1
Southern
HER2 Biology
4.4 kb -
p185 -
12.5 kb -
Slamon et al., Science 244: 707-712, 1989
(DNA)
(mRNA)
(protein)
HER2 amplification: HER2 / MPO ratio > 2.0
Uniform IHC staining
throughout each tumor
FISH
H & E
HER2 Biology
IHC
Slamon et al., Science 244:707-712, 1989; Pauletti et al., Oncogene 13:63-72, 1996
Southern Blot “Single Copy or Not-amplified”Overexpression: Actually HER2-amplified by FISH
HER2 Gene Amplification is Responsible for Overexpression
HER2 Biology
Fixation and Paraffin Embedding Result in
Decreased Antigenicity (Variable False-Negatives)
2 to 5-fold HER2 Amplified / Frozen IHC 2 to 5-fold Amplified / Fixed, Paraffin IHC
Immunohistochemistry in Formalin-Fixed, Paraffin-Embedded Tissues
Slamon et al., Science 244: 707-712, 1989
HER2 Protein Expression by IHC in Frozen Normal
Breast Tissues
HER2 IHC: Frozen tissue HER2 IHC: FFPEHematoxylin & Eosin
Press MF, Cordon-Cardo C, Slamon DJ. Oncogene 5: 953-962, 1990
HER2 IHC:
Frozen tissue, HER2
Immune serum
150x.
HER2 IHC: Pre-immune
serum control
1450x.
1450x. 1450x.
HER2 IHC:
Frozen tissue,
HER2 Immune
serum,
human breast
cancer with HER2
amplification
150x.
Q 1
HER2 Expression in Normal Adult and Fetal Epithelium:
Basal and Lateral, not Lumenal Immunostaining
Fetal Adult
Press MF, Cordon-Cardo C, Slamon DJ. Expression of the HER-2/neu Proto-oncogene in Normal Adult and Fetal Tissues. Oncogene 5: 953-962, 1990
Bronchus Bronchus
LiverBile Duct
Small Intestine
Small Intestine
Kidney Kidney Tubules
Endometrium Endometrium
Fallopian Tube Q 1
Breast Cancer: Basal and Lateral, but not Apical
Membrane Staining for HER2 Protein (IHC 3+)
HER2 gene amplification (FISH ratio = 11.70 / 1.45 = 8.07) Q 1
FISH-
FISH+
Time to Death (months)
Pro
babili
ty o
f surv
ival
9672480 24 1441200.0
0.50
0.75
1.00
Association with Poor Outcome in Node-Negative
Breast Cancer Patients
Press et al., Journal of Clinical Oncology 15:2894-2904, 1997
HER2 “positive”: FISH ratio = HER2 / CEP17 >2.0,
Average HER2 gene copy number >4.0
HER2 Gene Amplification by FISH
Q 2
Trastuzumab (Herceptin)
Monoclonal anti-HER2 antibody Humanized to:
– Avoid immunogenicity (95% human, 5% murine)
– Activate tumor-directed immune response
Possible mechanisms of action:
– Inhibition of abnormal signaling
– Interaction (synergy) with chemotherapy
– Enhancement of antibody-dependent cellular cytotoxicity (ADCC)
HER2 epitopes recognized by hypervariable murine
sequences
Human
IgG1
Carter et al. Proc Natl Acad Sci U S A. 1992;89:4285.
Slamon DJ et al, NEJM, 344:783-92, 2001.
MedianSurvival
(mo)Odds Ratio P Value
IHC+(2+/3+)
H + CT 25.10.80 0.05
CT 20.3
Pivotal Trial of Trastuzumab in Metastatic Breast Cancer: Association with Prolonged Overall Survival
0 10 20 30 40 50Months
0
0.2
0.4
0.6
0.8
1.0
Pro
bab
ility
of
surv
ival
Q 2
Breast Cancer International Research Group (BCIRG)-006 Trial
of Adjuvant Trastuzumab in Early Breast Cancer:
Disease Free Survival%
Dis
ea
se
Fre
e0
.50
.60
.70
.80
.91
.0
0 1 2 3 4 5
Patients Events
1073 192 AC->T1074 128 AC->TH
1075 142 TCH
81%
87%
86%
77%
83%
82%87%
93%
92%
HR (AC->TH vs AC->T) = 0.61 [0.48;0.76] P<0.0001
HR (TCH vs AC->T) = 0.67 [0.54;0.83] P=0.0003
Year from randomization
Slamon et al., NEJM, 2011
ChemoRx
ChemoRx
+ Trastuzumab
Approximately half of breast cancers were ER+ and these patients derived
significant benefit from trastuzumab treatment. Q 2
FDA-approved drugs for treatment of patients
with breast cancers having HER2 (aka ERBB2)
amplification / overexpression
• Trastuzumab
• Pertuzumab
• TDM1 (Ado-trastuzumab emtansine)
• Lapatinib
• Neratinib
Q 3
Press MF, Kim G,
Khoshchehreh MMK, Ma
Y, Slamon DJ. HER2
Testing in the Era of
Changing Guidelines, in
HER2-Positive Breast
Cancer, Edited by Sara
Hurvitz, pp 13-39, 2018
HER2 Overexpression Detection by IHC Negative or, 0+ 1+
2+ 3+
HER2 Immunohistochemistry (IHC)
IHC is a standard assay method in most anatomical pathology laboratories
which is easily performed and easily interpreted. Q 4
ASCO-CAP Guideline Testing Algorithm for
HER2 Testing by IHC
• 95% correlation required between HER2
status by FISH for IHC 0, 1+, and 3+. No targeted Therapy
Journal of Clinical Oncology 25: 118-145, 2007
01+3+
Treatment with HER2Targeted Therapy (e.g.trastuzumab)
No targeted therapy
2013 /
2014
2007
Algorithm for HER2 Testing by IHC
Changed from >10% to >30%
Changed from >30% back to >10%
95% concordance
required between
IHC 0, 1+, 3+ and
FISH to screen with
IHC
95%
concordance
NOT required;
Lab discretion
Fixation only with formalin for 6 to 48 hours
Fixation only with formalin for 6
to 72 hours
Above: Wolff et al., J Clin Oncol 25: 118-145, 2007; Below: Wolff AC et al., J Clin Oncol 31: 3997-4013, 2013. Q 5
Concordance between IHC and FISH:
Prevalence of HER2 Gene Amplification in each IHC Category, 2008 - 2014
HER2 Gene Amplification Rate (%) in Each IHC Staining Category by Study
IHC 0 (%) IHC 1+ (%) IHC 2+ (%) IHC 3+ (%) Number IHC Method Reference
0% 8.3% 22.9% 56.3% 661 Dako HercepTest (FDA) Rasmussen BB et al Acta Oncol., 2008
1.6% 29.1% 86.4% 697A0485 (Dako)
Grimm et al, AJCP, 2010
12.2% 66.6% 93.9% 175 3B5 antibody (LDT) Panjwani et al, Indian J Med Res., 2010
3.3% 57.9% 95.2% 100 Dako HercepTest (FDA) Tsuda et al, BMC Cancer, 2010*
0% 3.3% 15.2% 84.1% 200 4B5 antibody, LDT Lambein et al, J Clin Pathol., 2011
0% 3.2% 21.5% 91% 681 Dako HercepTest (FDA) Jorgenson JT, AJCP, 2011
12.8% 43.8% 97.8% 291 A0485 (Dako), LDT Bernasconi B et al, Br Ca Res Treat., 2012
0% 23% 38.8% 100% 216 CB11 antibody Martin V et al, Patholog Res Int., 2012
3.3% 7.1% 49.2% 88.4% 543 CB11 antibody Lee et al, Arch Med Res., 2012
0% 12.5% 76.5% 97.3% 125 Dako HercepTest (FDA) Kiyose et al, Pathol Int., 2012
2.4% 39.9% 98.1% 1437 Dako HercepTest (FDA) Vergara-Lluri ME et al, Mod Pathol, 2012*
9.6% 38.9% 87.2% 396 CB11 (Biogenix) Kokate P et al, Genetic Test Mol Biomark, 2012
2.6% 4.8% 28.1% 93.8% 950 A0485 (Dako), LDT Park S et al, Cancer, 2012
0% 1% 19% 92% 154 Dako HercepTest (FDA) Minot DM et al, AJCP, 2012
10% 5% 13% 69% 2546 CB11 (Ventana) Varga Z et al, BMC Cancer, 2013
0% 2.6% 29.4% 100% 150 4B5 (Ventana) (FDA) Lambein K et al., AJCP, 2013
9.4% 6.4% 13.5% 55.1% 628 A0485 (Dako), LDT Fasching P et al., BCRT, 2014
1.7% 3.3% 12.4% 81.1% 2590 Dako HercepTest (FDA) Schalper KA et al, Arch Pathol Lab Med, 2014
Less than 95% Concordance of IHC with FISH assay results.
Concordance between IHC and FISH:
Prevalence of HER2 Gene Amplification in each IHC Category, 2014 - 2018
HER2 Gene Amplification Rate (%) in Each IHC Staining Category by Study
IHC 0 (%) IHC 1+ (%) IHC 2+ (%) IHC 3+ (%) Number IHC Method Reference
0.8% 0.7% 5.8% 84.3% 1528 Dako HercepTest (FDA)Varga Z et al, PLoS One, 2015
1.5% 16.4% 98.9% 811 4B5 (Ventana)Green IF et al, Hum Pathol, 2015*
31.3% 50.5% 95.2% 174 A0485 (Dako) (LDT)Pu X et al, Pathol Res Pract, 2015
1% 0.6% 16.8% 49.1% 3605 Dako HercepTest (FDA) Morey AL, Pathology, 2016
5.6% 40.3% 100% 3144B5 rabbit, Ventana
(FDA)Overcast WB et al, Virchows Arch, 201682
5.8% 6.2% 36.0% 96.4% 3684B5 rabbit, Ventana
(FDA)Solomon JP et al, Am J Clin Pathol, 2017
0% 3.3% 23.5% 100% 1294B5 rabbit, Ventana
(FDA)Qi L, Biochem Biophys Res Commun, 2017*
4.2% 31.1% 93% 432 Dako HercepTest (FDA)Eswarachary V et al, J Clin Diagn Res, 2017
3.2% 37.0% 97.8% 498 Dako HercepTest (FDA)Furrer D et al, Anticancer Res, 2017*
2.5% 7.4% 31.3% 85.4% Averages by Studies with 4 IHC categories
3.9% 36.5% 91.5% Averages by Studies with 3 IHC categories
Less than 95% Concordance of IHC with FISH assay results.
Algorithm for HER2 Testing by IHC in 2018:
Unchanged from 2013 / 2014
2007, 2013/2014 and 2018 Guidelines largely ignore both the IHC 0/1+ false-negative and the IHC3+ false-positives
HER2 Gene Assessment by FISH
Ratio <2.0 Not Amplified(FISH-)
Ratio >2.0 Amplified(FISH+)
Key Features:• Probes
– Direct labeled– HER2
sequence (red) – Chrom 17
centromere (green)
• Interpretation– Signal
enumeration– Ratio of
HER2:Chr 17 signals
HER2 “positive”: FISH ratio = HER2 / CEP17 >2.0
Comparison of Six Different HER-2 Assays in HER2 Molecularly
Characterized Breast Cancers
Press et al., Journal of Clinical Oncology 20: 3095-3105, 2002
Frozen IHC
Amplification Level :
Northern
Western
> 1
0
5 -
10
2 -
5
1
Southern
FDA-Approved IHC Assays
FDA-Approved FISH Assays
Lab-Dev IHC AssaysR60
R60
10H8
10H8
Concordance with Known Molecular HER2 Status
97.4% 95.7% 89.7% 88.9%Molecular Status as
Determined by
Southern, Northern,
Western blots and
Frozen IHC
N = 117
Outcomes of Women with IHC0/1+ IHC and FISH-Positive
Invasive Breast Cancers
Fasching et al., 2014Q 4
Mass R, Press MF, et al.
Clinical Breast Cancer 6: 240-246, 2005.
Slamon DJ et al, NEJM, 344:783-92, 2001.
MedianSurvival
(mo)Odds Ratio P Value
IHC+(2+/3+)
H + CT 25.10.80 0.05
CT 20.3
Pivotal Trial of Trastuzumab in Metastatic Breast Cancer Demonstrates the Importance of HER2 Amplification for
Responsiveness
0 10 20 30 40 50Months
0
0.2
0.4
0.6
0.8
1.0
Pro
bab
ility
of
surv
ival
FISH+
H + CT 26.20.71 0.007
CT 20.0
IHC 2+/3+ but FISH-NegIHC 2+/3+ and FISH-Pos
Q 4
Wolff A, et al., Journal of Clinical Oncology, 31: 3997-4013, 2013.
Wolff A, et al., Arch of Pathol Lab Invest, 138: 241–256, 2014.
ASCO-CAP Guidelines: 2013 / 2014
Optimal ASCO-CAP Algorithm for HER2 Testing by FISH: HER2 probe with a control CEP17 probe
Wolff A, et al., JCO, 2013; Arch of Pathol Lab Invest, 2014.
*
Group 1 Group 2 Group 3 Group 4 Group 5
No Published Data in 2013/ 2014.
Q 5
Study Goals
• Determine the frequency of each ASCO-
CAP HER2 FISH group.
• Evaluate each ASCO-CAP FISH group for
association with HER2 overexpression
• Assess ASCO-CAP groups for association with
outcomes in the absence of trastuzumab and
with trastuzumab treatment.
Archives of Pathology and Laboratory Medicine 140: 1250-1258, 2016
Journal of Clinical Oncology 34 (29): 3518-3528, 2016 N = 10,468
N = 7,526
Patients Screened in Central
Lab by FISHN=10,468
HER2 Not Amplified
N=6199 (59.2%)HER2 Amplified
N=4269 (40.8%)
BCIRG-005
N=3298BCIRG-006
N=3222
BCIRG-007
N=263
Arm 1. AC-T
N=1649Arm 2. TAC
N=1649
Arm 1. AC-T
N=1073Arm 2. ACTH
N=1074
Arm 3. TCH
N=1075
ASCO-CAP
ISH Group 5. N=3,079
ASCO-CAP
ISH Group 4. N=183
ASCO-CAP
ISH Group 3. N=16
ASCO-CAP
ISH Group 2. N=52
ASCO-CAP
ISH Group 1. N=3,321
Screening of Breast Cancers by BCIRG / TRIO Central
Laboratory for HER2 Status: Specimen Accountability
JAMA Oncology, 2019
Screening of Breast Cancers by BCIRG / TRIO Central
Laboratory for HER2 Status: Specimen Accountability
Journal of Clinical Oncology 34 (29): 3518-3528, 2016
Assessment of HER2 by FISH According to 2014
ASCO-CAP Guidelines by Group
Group Description of
FISH category
No. of
Cases
Overall
%
No. of
Cases
Overall
%
1 Ratio >2.0,
HER2 average >4.01328 17.7% 4269 40.8%
2 Ratio >2.0,
HER2 average <4.0 31 0.4% 71 0.7%
3 Ratio <2.0,
HER2 average >6.0 48 0.6% 55 0.5%
4 Ratio <2.0,
HER2 average >4.0,
<6.0
345 4.6% 432 4.1%
5 Ratio <2.0,
HER2 average <4.0 5774 76.7% 5641 53.9%
Totals 7526* 100% 10468 100%
*86 cases (1.1%) with HER2 Genomic Heterogeneity were excluded.
Consultation Study CIRG Trials Study
Q 5
ASCO-CAP FISH Groups: Comparison of HER2 Gene / CEP17 Status (FISH) with
HER2 Protein Expression (IHC)
Journal of Clinical Oncology 34 (29): 3518-3528, 2016
*
ASCO-CAP FISH Groupings Compared with HER2 Protein
by IHC Scores
ASCO-
CAP
Group
HER2-to-
CEP17
Ratio
Average
HER2
number /
cell
IHC 0
N (%)
IHC 1+
N (%)
IHC 2+
N (%)
IHC 3+
N (%)
Totals
(%)
P-value**
Group 1 >2.0 >4.0 240 (11.8%) 264 (12.9%) 571 (28.0%) 965 (47.3%) 2,040 <0.0001
Group 2 >2.0 <4.0 24 (68.6%) 8 (22.9%) 3 (8.6%) 0 (0%) 35 0.0007
Group 3 <2.0 >6.0 5 (55.6%) 2 (22.2%) 1 (11.1%) 1 (11.1%) 9 0.3881
Group 4 <2.0 >4.0, <6.0 105 (78.4%) 21(15.7%) 7 (5.2%) 1 (0.7%) 134 <0.0001
Group 5 <2.0 <4.0 1,988 (94.1%) 114 (5.4%) 10 (0.5%) 1 (0.05%) 2,113 <0.0001
*IHC = immunohistochemistry; when data from both HER2 immunohistochemical assays, 10H8 and HercepTest, were
available the HercepTest assay result was used.
**P-value based on chi-square test for goodness of fit test of the hypothesis of equal proportions in each of the 4 IHC
categories
Journal of Clinical Oncology 34 (29): 3518-3528, 2016
ASCO-CAP Algorithm for HER2 Testing by FISH: 2018
Wolff A, et al., JCO, 2018
No published data related to “problematic issues” with chromosome 17
“alternative control probes” (e.g. TP53, D17S122, SMS, RARA, TOP2A) for ISH Q 7
Evaluation of FISH Group 2
91%* 9%* 0%*
Wolff A, et al., JCO, 2018
*Press MF et al., JCO, 2016
Comparison of HER2 Ratio and Average HER2 Gene Copy Number by ASCO-CAP Groupings with Clinical Outcomes in BCIRG-006 Trial
HER2
FISH
Ratio
HER2
copies
per cell
No. of
subjects
DFS
Control (events/no. of
subjects)
DFS
Trastuzu
mab(events/
number of
subjects)
DFS, HR
DFS
(95%
CI)*
DFS, P for
Log Rank
test*
OS
Control (events/no.
of
subjects)
OS
Trastzu
mab
OS, HR
(95% CI)*
OS P for
Log
Rank
test OS*
ASCO-CAP
FISH Group
Ratio
>2.0
<4.0 46 4 / 18 6 / 28 1.10
(0.31,
3.89)
0.8860 2 / 18 4 / 28 3.15
(0.35,
28.63)
0.2839 Group 2
>4 3109 251 /
1031
391 /
2078
0.71
(0.60-
0.83)
<.0001 138 /
1031
202 /
2078
0.69
(0.55-
0.85)
0.0006 Group 1
Total: 3155
NOTE. The HRs are for Trastuzumab treatment arms compared with Control chemotherapy only arm. There were too few patients (n =
5) accrued to BCRIG-006 with a HER2 FISH ratio <2.0 and >6.0 average HER2 gene copy number/tumor cell for analysis of the HR.
Abbreviations: BCIRG, Breast Cancer International Research Group; CAP, College of American Pathologists; DFS, disease-free
survival; HER2, human epidermal growth
factor receptor 2; HR, hazard ratio; OS, overall survival.
*Trastuzumab-containing treatment arms compared with control (chemotherapy alone) treatment arm.
Press et al., Journal of Clinical Oncology, 2016.
Evaluation of FISH Group 3
78%* 11%* 11%*
Wolff A, et al., JCO, 2018
*Press MF et al., JCO, 2016
Comparison of HER2 Gene Amplification Status with HER2 Protein
Expression by a Laboratory-Developed IHC Assay (10H8-IHC) in
ASCO-CAP Group 3 Patients Randomized to a BCIRG Trial.
ASCO-CAP
Group (Ratio
<2.0 and
Average
HER2 copies
>6.0)
HER2 BCIRG
FISH Status
Mean of
average
HER2 copy
numbers
IHC 0 IHC 1+ IHC 2+ IHC 3+ Totals
Group 3A Amplified Average
16.38
1 (17%) 0 (0%) 3 (50%) 2 (33%) 6 (24%)
Group 3N Not
Amplified
Average
7.43
8 (42%) 9 (47%) 2 (11%) 0 (0%) 19 (76%)
9 9 5 2 25 (100%)
There is a significant difference between Group 3A and Group 3N in terms of IHC staining with 83% of
Group 3A IHC 2+/3+ compared with 89% of Group 3N that were IHC 0/1+ (p=0.002, Fisher’s exact test).
Press et al., Journal of Clinical Oncology, 2016.
Minority of ASCO-CAP FISH Group 3 breast cancers (our “Group 3A”)
show HER2 gene amplification and HER2 protein overexpression
HER2 : CEP17 = 1.47
HER2CEP17 CEP17
SMS RARA
Press et al., Arch Pathol Lab Med, 2016
IHC 3+ (HercepTest)
HER2 = 23.2 / cell
CEP17 = 15.75 / cell
HER2
RARA = 2.55 / cell
SMS = 1.85 / cell
HER2 : RARA =
23.2 / 2.55 = 9.1
HER2 : SMS =
23.2 / 1.85 = 12.54
Evaluation of FISH Group 4
Wolff A, et al., JCO, 2018
94%* 5%* <1%*
*Press M, JCO, 2016Q 6
Comparison of HER2 Ratio and Average HER2 Gene Copy Number
by ASCO-CAP Groupings with Clinical Outcomes in BCIRG-005 Trial
HER2 FISH
(HER2 /
CEP17)
Ratio
HER2
copies per
cell
No. of
subjects
DFS
(no. of
events)
OS
(no. of
events)
DFS,
HR (95% CI)
and P-values
for logrank
test*
OS,
HR and P-
values for
logrank
test*
ASCO-
CAP FISH
Group
Ratio
<2.0
4.01-6.0 176 51 30 0.923(0.697-1.224)
P=0.5795
0.878(0.609-1.267)
P=0.4872
Group 4
Ratio
<2.0
<4.0 3079 971 606 1.0
(reference)
1.0
(reference)
Group 5
The hazard ratios are for ASCO-CAP Group 4 compared with ASCO-CAP Group 5 taken as
the reference in the BCIRG-005 (HER2-not-amplified) breast cancer trial.
OS = overall survival
DFS = disease-free survival
Press et al., Journal of Clinical Oncology, 2016. Q 6
Resolution of “HER2 (FISH) Equivocal” Breast Cancers (ASCO-
CAP Group 4) according to 2013 / 2014 ASCO-CAP Guidelines
through the use of Chr 17 Alternative Control Probes
Smith-Magenis syndrome
Alternative Control Probes for HER2 Equivocal
Breast Cancers
J Clin Oncol 29:4168-4174, 2011
“Among the cases with mean HER2 copy number of 4 to 6, 41 (47.7%) of 86
had their HER2 gene status upgraded from nonamplified to amplified”
HER2 copies / any Alt Control >2.0
Use of Chr 17 Alternative Control Probes for Evaluation of
“HER2 (FISH) Equivocal” Breast Cancers
Mayo Clinic:
JCO, 34: 3502-3510, 2016
Cleveland Clinic:
Cancer 123: 2230-2239,
2017.
M. D. Anderson Cancer Ctr:
Cancer, 123: 1115-1123,
2017.
Of 405 patients initially considered FISH-
equivocal (ratio <2.0 with HER2 signal >4.0,
but <6.0, use of an alternative chromosome
17 probe reassigned 212 patients to FISH-
positive: (52.3%).
57 HER2 “equivocal” to 35
“amplified” with D17S122: 61%
73 HER2 “equivocal” to 38
“amplified” with D17S122: 52%
38 of 73 (52%) “HER2 equivocal” breast cancers were “re-classified” as “amplified”
(Donaldson AR, et al. Cancer, 2017)
“We evaluated 345 patients with node positive disease in a blinded fashion (Table 1).
Of these, 101 (27%) had evidence of HER-2/neu amplification. Univariate (as well as
multivariate) survival analysis showed amplification of the HER-2/neu gene to be a
significant predictor of both disease-free survival and overall survival for these patients
(Table 1).” (Slamon et al., Science 244: 707-712, 1989) NOTE: MPO was the internal
control gene for assessment of amplification, i.e. a HER2-to-MPO ratio >2.0.
“HER-2/neu amplification was determined by the ratio of the HER-2/neu signal
relative to the single copy p53 signal.” “The overall amplification rate was 33%.”
“Amplification of the HER-2/neu gene did not correlate with either disease-free or
overall survival in univariate or multivariate analyses.” (Clark and McGuire, Cancer
Res. 51, 944-948, 1991)
Importance of an Appropriate Internal Control for Assessment of
Amplification
Distribution of average HER2 gene copies and HER2 FISH ratios among breast cancers successfully screened for enrollment into BCIRG trials from
2000 to 2004
Press et al., JCO, 2016
Relative Copy Number of HER2 / ERBB2 and Genomic Sites used as Alternative
Controls to determine HER2 Status by FISH (METABRIC SNP array data; N = 1980)
Re
lative
Ge
ne
Co
py N
um
be
r
Ga
in
0 Lo
ss
AmplGain
HER2 / ERBB2
LIS1TP53D17S122RAI1SMSERBB2RARA–TOP2A
Press MF, Seoane JA, Curtis C et al. JAMA Oncology, 2019
Chromosome 17 Regional Gene Copy Gains / Losses based on GISTIC among Alternative Control
Genomic Sites Compared to HER2/ERBB2 Gene Copy Gains / Losses in the METABRIC Cohort (N = 1915)
Press MF, Seoane JA, Curtis C et al. JAMA Oncology, 2019
Patients Screened in Central
Lab by FISH
N=10,468
HER2 Not Amplified
N=6199 (59.2%)HER2 Amplified
N=4269 (40.8%)
BCIRG-005
N=3298
BCIRG-006
N=3222
BCIRG-007
N=263
Arm 1. AC-T
N=1649Arm 2. TAC
N=1649
Arm 1. AC-T
N=1073Arm 2. ACTH
N=1074
Arm 3. TCH
N=1075
ASCO-CAP
ISH Group 5.
N=3,079
ASCO-CAP
ISH Group 4.
N=183
ASCO-CAP
ISH Group 3.
N=16
ASCO-CAP
ISH Group 2.
N=52
ASCO-CAP
ISH Group 1.
N=3,321
FISH-
negative.
N=100
“FISH-
Equivocal”
N=100
Evaluation of HER2-Equivocal and HER2-Not-Amplified Breast Cancers by
FISH: Specimen Accountability
JAMA Oncology, 2019
Outcomes for ASCO-CAP Group 4 (HER2-Equivocal) and
ASCO-CAP Group 5 (HER2-not-amplified) Breast Cancer
Patients: DFS and OS.
Disease Free Survival by group (group 4=HER2-Equivocal, group 5=HER2-not-amplified)
Prop Disease Free
0.0
0.2
0.4
0.6
0.8
1.0
0 24 48 72 96 120 144
Months
Cohort Patients Events
Group 4 100 36
Group 5 100 33
100 83 71 57 53 38 1
100 84 72 60 52 44 1 Group 5
Group 4
Number Disease Free
Overall Survival by group (group 4=HER2-Equivocal, group 5=HER2-not-amplified)
Proportion Alive
0.0
0.2
0.4
0.6
0.8
1.0
0 24 48 72 96 120 144
Months
Cohort Patients Events
Group 4 100 22
Group 5 100 20
100 87 79 71 65 46 1
100 94 81 67 60 53 1 Group 5
Group 4
Number Alive
Disease-Free Survival of ASCO-CAP FISH
Group 4 (HER2-Equivocal) Compared to
ASCO-CAP FISH Group 5 (HER2-negative)
Overall Survival of ASCO-CAP FISH Group
4 (HER2-Equivocal) Compared to ASCO-CAP FISH Group 5 (HER2-negative)
JAMA Oncology, 2019
Overall Survival for ASCO-CAP Group 4 (HER2-Equivocal) and ASCO-CAP Group 5 (HER2-not-amplified) Breast Cancer
Patients by HER2 / Alternative Probe RatiosOverall Survival by HER2/D17S122 ratio
HER2-Equivocal
Proportion Alive
0.0
0.2
0.4
0.6
0.8
1.0
0 24 48 72 96 120 144
Months
Cohort Patients Events
<2 70 15
>=2 30 7
70 62 56 52 47 32 1
30 25 23 19 18 14 >=2
<2
Number Alive
ASCO-CAP FISH Group 4 (HER2-Equivocal):
OS for HER2 / D17S122 Ratios >2.0 versus
Ratios <2.0
Overall Survival by HER2/D17S122 ratio
HER2-not-amplified
Proportion Alive
0.0
0.2
0.4
0.6
0.8
1.0
0 24 48 72 96 120 144
Months
Cohort Patients Events
<2 89 17
>=2 11 3
89 84 74 62 55 48 1
11 10 7 5 5 5 >=2
<2
Number Alive
ASCO-CAP FISH Group 5 (HER2-negative):
OS for HER2 / D17S122 Ratios >2.0 versus
Ratios <2.0
JAMA Oncology, 2019
>2.0 >2.0
<2.0<2.0
Q 6
Overall Survival for ASCO-CAP Group 4 (HER2-Equivocal) and ASCO-CAP Group 5 (HER2-not-amplified) Breast Cancer
Patients by HER2 / Alternative Probe RatiosOverall Survival by HER2/SMS Ratio
HER2-Equivocal
Proportion Alive
0.0
0.2
0.4
0.6
0.8
1.0
0 24 48 72 96 120 144
Months
Cohort Patients Events
<2 39 9
>=2 61 13
39 34 28 26 24 19 1
61 53 51 45 41 27 >=2
<2
Number Alive
Overall Survival by HER2/SMS Ratio
HER2-not-amplified
Proportion Alive
0.0
0.2
0.4
0.6
0.8
1.0
0 24 48 72 96 120 144
Months
Cohort Patients Events
<2 63 10
>=2 37 10
63 61 52 40 35 31 1
37 33 29 27 25 22 >=2
<2
Number Alive
ASCO-CAP FISH Group 4 (HER2-Equivocal):
OS for HER2 / SMS Ratios >2.0 versus
Ratios <2.0
ASCO-CAP FISH Group 5 (HER2-negative):
OS for HER2 / SMS Ratios >2.0 versus
Ratios <2.0
JAMA Oncology, 2019
>2.0
>2.0<2.0
<2.0
Q 6
Criteria for Evaluation of Heterozygous Deletions at Alternative Control
Genomic Sites on Chromosome 17 by FISHChromosome
17 Arm
Gene /
Locus
Ratio Interpretation Ratio Interpretation
p-arm SMS<0.75a
SMS with
heterozygous deletion
relative to RARA
>1.25dRARA with
heterozygous deletion
relative to SMSq-arm RARA
p-arm TP53 <0.75b
TP53 with
heterozygous deletion
relative to TOP2A
>1.25eTOP2A with
heterozygous deletion
relative to TP53q-arm TOP2A
p-arm D17S122 <0.75c D17S122 with
heterozygous deletion
relative to HER2>1.25f
HER2 with
heterozygous deletion
relative to D17S122 q-arm HER2
SMS, Smith-Magenis syndrome locus; RARA, retinoic acidreceptor-alpha gene; TP53, tumor protein 53 tumorsuppressor gene; TOP2A, topoisomerase-II-alpha gene;D17S122: the 17p-arm genomic locus which is duplicated inCharcot-Marie-Tooth disease; HER2, human epidermal growthfactor receptor 2 gene.
CE
P17
JAMA Oncology, 2019
HER2 Equivocal by FISH: Heterozygous
Deletion of SMS relative to RARA
HER2 / CEP17 = 4.42 / 2.58 = 1.72
HER2 CEP17
HER2 / RARA = 4.42 / 4.40 = 1.00
HER2 / SMS = 4.42 / 2.05 = 2.15
RARA SMS
HER2 IHC by 10H8 assay: IHC 0
HER2 IHC by Dako HercepTest: IHC 1+Press et al. JAMA Oncology, 2019
Comparison of FISH Groups with FDA-Approved Status, ASCO-CAP
Guidelines Recommendations, HER2 Protein Expression by IHC, and
Associations with Outcomes in BCIRG Clinical Trials
Grp Ratio Average
HER2
% FDA ASCO-
CAP
2014
2018
HER2
Protein
Progn
BCRIG-
005
Trast
Resp
BCIRG-
006
BCIRG /
TRIO
1 > 2.0 > 4.0 40.8 Ampl ISH + Overexp - Signific
Improv
Amplified
2 > 2.0 < 4.0 0.7 Ampl ISH+
IHC
Low Ex - Not sig Not Ampl
3 < 2.0 > 6.0 0.5 Not Am ISH+
IHC
Mixed Indeter Indeter Mixed
4 < 2.0 > 4.0,
<6.0
4.1 Not Am ISH?
IHC
Low Ex Not Worse - Not Ampl
5 < 2.0 < 4.0 53.9 Not Am ISH - Low Ex Reference - Not Ampl
Press et al., Journal of Clinical Oncology, 2016; JAMA Oncology, 2019.
Conclusions • Development of Companion Diagnostics for clinical
trials and patient management are complex regulatory as well as research issues.
• In spite of three decades of research, HER2 testing for selection of patients to targeted therapies remains controversial. – Implementation of new ASCO-CAP guidelines for HER2 FISH
testing result in no changes for approximately 90%-95% of cases.
– Changes in ASCO-CAP guidelines for “groups 2-4” will result in potential disagreements for approximately 5% of cases.
• The use of Chr 17 alternative control genes, especially p-arm genes, as alternative controls to assess HER2 gene status may lead to “false-positive” ratios by FISH due to heterozygous deletion of chromosome 17p-arm genomic sites.
Acknowledgements• USC (Press Laboratory)
– Yanling Ma, MD
– Simon Davenport
– Armen Gasparyan
– Roberta Guzman
– Olivia Franco
– Angela Santiago
– Ivonne Villalobos
– Bin Xie, MD, PhD
– Caihong Xia, PhD*
– Michael Gordon, PhD*
– Brandon Li, MD*
– Mariana Keshmeshian, PhD*
– Jinha Park, MD, PhD*
– Melinda Epstein, PhD*
– Anamaria Ioan, MD, PhD *
– Jian-Yuan Zhou, MD*
• Stanford U – Christina Curtis, PhD
– Jose Seoane, PhD
• Memorial Sloan Kettering – Malcolm Pike, PhD
• M. D. Anderson Cancer Center – Adel El-Naggar, MD
– Lovell Jones, PhD
• City of Hope National Medical Center– Leslie Bernstein, PhD
• Ventana Medical Systems, Inc. – Michael Barnes, MD – Leigh Ann Henricksen, PhD – Larry Morrison, PhD
• Abbott-Vysis, Inc. – Kerry Flom, PhD– Steven Seelig, MD, PhD
• Genentech, Inc. – Robert Mass, MD– Pam Klein, MD
• GlaxoSmithKline– Cathy Ellis, PhD – Maria Koehler, MD, PhD– Anne Marie Martin, PhD
• Caris Life Sciences, Inc. – Wenhsiang Wen, MD, PhD* – Wangjuh (Sting) Chen, PhD
• Cepheid, Inc. – Michael Bates, MD – Natalie C. Wu, PhD – Wendy Wong– Kenneth E. Ho – Victor C. Chu, PhD – Analiza Rizo– Jodi M. Weidler, PhD
The Women who participated in the Clinical Trials
• UCLA– Dennis Slamon, MD, PhD
– Richard Finn, MD
– Gottfried Konecny, MD
– Zev Wainberg, MD
– Sara Hurvitz, MD
• University of Hamburg– Guido Sauter, MD
– Martina Mirlacher
– Tobias Grob, MD
• Cancer International Research Group / TRIO – Valerie Bee
– Henry Taupin
– Karen Afenjar
• Erlangen University (Bavarian Breast Cancer Research
Group)– Peter Fasching, MD
• Grant Support:– NCI – California Breast Cancer Research
Program – DOD Breast Cancer Research Program – Breast Cancer Research Foundation– Tower Cancer Research Foundation – Adelson Medical Research Foundation